The Regulatory Role Of CAF-derived IL-6-induced Osteopontin In The Growth And Metastasis Of Oral Squamous Cell Carcinoma

Background&Purpose:In our previous study,secreted phosphoprotein 1(SPP1)gene was found to be significantly upregulated in oral squamous cell carcinoma(OSCC).The osteopontin(OPN)protein is encoded by SPP1 gene,which is a secreted chemokine-like protein and plays an important role in the growth and metastasis of a variety of malignant tumors.However,it is still unclear how tumor stroma regulates the expression level of neoplastic OPN and how the multifaceted OPN modulates the biological characteristics of OSCCs.In this study,we will try to detect the mRNA expression levels of SPP1 in OSCC,to analyze the correlations between the SPP1 mRNA levels and the clinicopathologic parameters of OSCC patients,to analyze the effect and the underlying mechanism of OPN on OSCC progression,to explore how tumor stroma modulates the OPN expression in OSCC cells and to further investigate the predictive efficacy of SPP1 in the prognosis of OSCC.Methods:The expression levels of SPP1/OPN were detected in OSCC tissues using immunohistochemical staining and quantitative real-time PCR.The correlations between SPP1 mRNA expression and the clinicopathologic characteristics of OSCC patients were then analyzed.Small interfering RNA or SPP1-expressiong plasmid transfection was performed to knock down or upregulate SPP1 expression in OSCC cells.Cell proliferation assay,colony formation assay,transwell migration and invasion assay were adopted to assess the effects of OPN on the biological behaviors of OSCC cells.A co-culture system was implemented to study the mechanism by which cancer associated fibroblasts(CAFs)modulated the OPN expression in OSCC cells Co-immunoprecipitation analysis was carried out to determine the endogenous interaction between OPN protein and integrin ?v?3.An intracellular signaling pathway antibody array was applied to analyze the detailed mechanism for OPN-mediated alterations to the cellular biological behaviors of OSCCs.Furthermore,a xenograft tumor assay and an experimental metastasis assay were performed to confirm the in vivo tumor-promoting role and the potential therapeutic value of OPN-targeting therapy in OSCCs.Results:SPP1 mRNA expression was significantly upregulated in OSCC tissues,and the increased SPP1 mRNA levels were markedly correlated with larger tumor size,lymph node metastasis,advanced TNM stage and poor prognosis of the patients with OSCCs.The immunohistochemical staining results revealed that OPN was primarily upregulated in cancer cells,especially in cancer cells of the edge of cancer nests Meanwhile,the expression levels of SPP1 gene or OPN protein were obviously upregulated in OSCC cells compared with that in normal oral epithelial cells.In cancer cells,the elevated neoplastic OPN activated the NF-?B signaling pathway by binding to integrin ?v?3.The activated NF-?B signaling subsequently triggered the transcription of the downstream genes,such as MMP2,MMP9,uPA and ICAM-1,and then regulated the growth,migration and invasion of OSCC cells.Moreover,the interaction between OSCC cells and CAFs was proved to promote the OPN expression in OSCC cells.Further mechanical analysis indicated that IL-6 secreted by CAFs was identified as a upstream molecule which accelerated the expression of neoplastic OPN by stimulating the transcriptional activity of STAT3 in OSCC cells.As such,CAF-secreted IL-6 could promote the malignant transformation of OSCCs through OPN-?v?3-NF-?B signaling pathway.More importantly,inhibiting the functions of OPN and IL-6 at the same time could significantly inhibit the growth and metastasis of OSCCs.In addition,compared with single molecule alone,the combination of SPP1 and IL-6 had a more promising predictive value in the prognosis of OSCC patients.Conclusions:This study identified a novel modulatory mechanism that CAF-secreted IL-6 regulated the biological behaviors of OSCC cells through OPN-NF-?B signaling pathway,and suggested that the combination of SPP1 and IL-6 might be a promising prognostic predictor as well as a potential therapeutic target for OSCCs.